National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
Biotechnol Lett. 2020 Apr;42(4):625-632. doi: 10.1007/s10529-020-02809-0. Epub 2020 Jan 20.
To set up an efficient gene editing system in Bacillus thuringiensis (Bt) using CRISPR-Cas9 by demonstrating deletion of chromosomal and plasmid genes.
CRISPR-Cas9 from Streptococcus pyogenes was found to function in Bt cells, resulting in DNA cleavage that is lethal to the cells. The system was assessed for its ability to mediate gene editing by knock-out of the protease genes nprA (neutral protease A) and aprA (alkaline protease A). Gene editing was not detected when the Bacillus-derived pBCX was used to carry CRISPR-Cas9 elements and a DNA repair template. When the Cas9 promoter was replaced with the sporulation-specific promoter cyt2A, a Bt ∆nprA clone was obtained, but this plasmid construct did not give reproducible results. Bt ∆nprA ∆aprA and Bt ∆aprA deletion mutants were finally generated when the Lactobacillus plantarum-derived plasmid pLPPR9 was used, likely due to its lower copy number reducing Cas9 toxicity. Only three to four clones each needed to be screened to identify the desired gene-modified mutants. Conversely, efficient editing of the plasmid vip3A gene required the use of pBCX and longer homology sequences for the repair template.
Capitalizing on the differential impact of plasmid copy number and homology arm length, we devised distinct yet simple and efficient approaches to chromosomal and plasmid gene deletion for Bt that condense the screening process, minimize screening, and facilitate multiple consecutive gene editing steps.
通过证明染色体和质粒基因的缺失，在苏云金芽孢杆菌（Bt）中建立高效的 CRISPR-Cas9 基因编辑系统。
发现来自酿脓链球菌的 CRISPR-Cas9 在 Bt 细胞中起作用，导致对细胞致命的 DNA 断裂。该系统通过敲除蛋白酶基因 nprA（中性蛋白酶 A）和 aprA（碱性蛋白酶 A）来评估其介导基因编辑的能力。当使用源自芽孢杆菌的 pBCX 携带 CRISPR-Cas9 元件和 DNA 修复模板时，未检测到基因编辑。当 Cas9 启动子被芽孢杆菌特异性启动子 cyt2A 取代时，获得了 Bt ∆nprA 克隆，但该质粒构建体没有产生可重复的结果。当使用来源于植物乳杆菌的质粒 pLPPR9 时，最终生成了 Bt ∆nprA ∆aprA 和 Bt ∆aprA 缺失突变体，可能是由于其较低的拷贝数降低了 Cas9 的毒性。只需筛选三到四个克隆即可鉴定出所需的基因修饰突变体。相反，有效编辑质粒 vip3A 基因需要使用 pBCX 和更长的同源序列作为修复模板。
利用质粒拷贝数和同源臂长度的差异影响，我们设计了针对 Bt 的截然不同但简单有效的染色体和质粒基因缺失方法，浓缩了筛选过程，减少了筛选，并促进了多个连续的基因编辑步骤。
